This proposal is based on three features of breast cancer. First, local stroma can influence the course of mammary tumorigenesis, although it is not yet clear just how it does so. Second, mammary cancer typically develops in older individuals. Third, estrogens somehow influence progress of the disease. The hypothesis to be tested is that stromal cell populations manifest certain changes as they age and senesce, and that these changes cause them to inappropriately stimulate adjacent epithelium. Such inappropriate stimulation is a necessary component of early tumor development. This project is designed to begin defining the relevant changes in populations of qed stromal (fibroblastic) cells. The first specific aim is to identify and characterize the bioactive molecules produced and secreted by aged stromal cells in culture. The studies will be focused on the epidermal growth factor and transforming growth factor beta families of peptides, both of which are now strongly implicated in regulation of neoplastic mammary growth. Growth factor production will be screened using immunological procedures, column chromatography, Northern blotting, and peptide sequencing if warranted. Changes in growth factor production in response to estrogens/antiestrogens will also be evaluated. The second specific aim will be to begin characterizing aged stromal cells to define why they behave abnormally. Stromal cell populations that prove to be growth-stimulatory in culture will be replaced into mammary glands in vivo to see if they retain their bioactivity towards epithelium. Stromal cell populations will be screened by immunohistochemistry using cell type-specific markers to define more accurately just what population(s) of cells is being studied. Characteristics of binding of ligands (EGF/TGFalpha, TGFbeta, estrogens) that could alter stromal cell function will be measured and compared among aged and control cell populations. Growth characteristics of stromal cells in response to these regulatory ligands will be assessed. The project is designed to clarify how the stromal microenvironment contributes at the cell level to hyperplasia during early stages of mammary tumorigenesis in older mammals.